Inhibiting T regulatory cells: T regulatory cells are known to be inhibitors of the immune response. One strategy to enhance immune response to cancer is by targeting the T regulatory cells. To be able to do that we embarked on studying the intrinsic signal transduction pathway of regulatory T cells (Tregs) with the intention that it may be targeted by small molecules. Using FoxP3 as a marker of mouse regulatory T cells instead, we found that the PI3K-Akt-mTOR pathway is different between the CD4+FoxP3+ regulatory cells and conventional CD4+ cells when stimulated with CD3 CD28 antibodies and/or Interleukin-2(IL-2).Two PI3K-Akt inhibitors, Wortmannin (WN) and LY294002 (LY) can significantly inhibit the phosphoralytion of P70S6 in Tregs more than in the conventional CD4 T cells counterpart. In vivo administration of WN and LY can specifically deplete Tregs in nave and tumor bearing mice. Our data showed that CD4+FoxP3+ cells are more sensitive to the PI3K inhibitors, and it's possible to manipulate this population of cells in vivo by administration of WN and LY (manuscript is in preparation). We are now, dissecting these differences in more elaborate fashion and we are trying testing the efficacy of this method in enhancing vaccine effect.-Enhancement the cancer vaccine by down regulation of negative signals: One of the major barriers to a successful anti-tumor vaccine is tolerance of T cells to tumor antigen. We are approaching the problem with two aims. One is targeting molecules that delivery the negative signal to T cell and DCs, such as CTLA-4, PD-1 and CD223. Our preliminary data showed that anti-CTLA and/or anti-PD1 could also enhance cancer vaccine and lead tumor rejection using tumor bearing mice. Second approached is to utilizing the small molecular inhibitor to enhance cancer vaccine. -Enhancing memory immune response: We have studied anti-CD40, as vaccine adjuvant in the generation of specific CD8 memory T cell response and long term protection of tumor rechallenge. Also, the data identifies the mechanism by which CD40 mediates the generation of high avidity CD8+ memory and show the different role that IL-15 plays in the generation of high versus low avidity T cells. Finally, the data suggests that local administration of anti-CD40 or soluble CD40 ligand have potential to be incorporated into vaccine therapy in promoting early and lasting immune responses to irradiate tumor and virus infection. Manuscript is submitted. To further understand the role of CD40 in generation of memory CD8+ T cell response, we are going to study the role of CD4 in generation of CD40 mediated memory response.